This proposal aims at providing basic knowledge relevant to metalloprotein design, the pre-determined placement and alteration of metal centers in proteins. A primary goal is to define the structural basis for the binding and activity of biologically important metal ions in metalloproteins. First, I will use existing metalloprotein structures as templates for the design of metal binding mutants in green fluorescent protein (GFP). These sites will be positioned to shift GGFP fluorescence upon metal ion binding. Successful metal ion binding GFP mutants will be characterized biochemically and crystallographically. Second, I will test the impact of the protein neighborhood by random mutagenesis of sites neighboring the bound metal ion. These random mutants will be selected using the power of cell sorting by fluorescent shifts mediated by metal ion binding to GFP. Third, successful designs will be tested using fluorescent microscopy as possible probes of specific metal ions within eukaryotic cells. Overall, this work uses the synergistic techniques of molecular biology, x-ray crystallography, and cell biology to examine metal ion binding specificity and affinity by proteins.